Following Structural Changes by Thermal Denaturation Using Trapped Ion Mobility Spectrometry-Mass Spectrometry

The behavior of biomolecules as a function of the solution temperature is often crucial to assessing their biological activity and function. While heat-induced changes of biomolecules are traditionally monitored using optical spectroscopy methods, their conformational changes and unfolding transitions remain challenging to interpret. In the present work, the structural transitions of bovine serum albumin (BSA) in native conditions (100 mM aqueous ammonium acetate) were investigated as a function of the starting solution temperature (T similar to 23-70 degrees C) using a temperature-controlled nanoelectrospray ionization source (nESI) coupled to a trapped ion mobility spectrometry-mass spectrometry (TINS-MS) instrument. The charge state distribution of the monomeric BSA changed from a native-like, narrow charge state ([M + 12H](12+) to [M + 16H](16+) at similar to 23 degrees C) and narrow mobility distribution toward an unfoldedlike, broad charge state (up to [M + 46H](46+) at similar to 70 degrees C) and broad mobility distribution. Inspection of the average charge state and collision cross section (CCS) distribution suggested a two-state unfolding transition with a melting temperature T-m similar to 56 +/- 1 degrees C; however, the inspection of the CCS profiles at the charge state level as a function of the solution temperature showcases at least six structural transitions (T1-T7). If the starting solution concentration is slightly increased (from 2 to 25 mu M), this method can detect nonspecific BSA dimers and trimers which dissociate early (T-d similar to 34 +/- 1 degrees C) and may disturb the melting curve of the BSA monomer. In a single experiment, this technology provides a detailed view of the solution, protein structural landscape (mobility vs solution temperature vs relative intensity for each charge state).